Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR
The E. coli O157: H7 is an enterohemorrhagic bacteria producing Shiga toxin. It can trigger diseases such as hemolytic uremic syndrome (HUS) and hemorrhagic colitis. It has been found associated with outbreaks of foodborne illness. Therefore, it is important to ensure their absence in the production...
- Autores:
-
Tere Peña, Claudia Patricia
- Tipo de recurso:
- Work document
- Fecha de publicación:
- 2020
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/77923
- Acceso en línea:
- https://repositorio.unal.edu.co/handle/unal/77923
- Palabra clave:
- 660 - Ingeniería química
570 - Biología
664 - Tecnología de alimentos
628 - Ingeniería sanitaria
E. coli O157: H7
Absolute quantification
Reference material
Method validation
Digital PCR
E. coli O157:H7
PCR digital
Cuantificación absoluta
Material de referencia
Validación de método
- Rights
- openAccess
- License
- Atribución-NoComercial-SinDerivadas 4.0 Internacional
| id |
UNACIONAL2_1e4c89746e29f7525c9a4fd0ea7ce3bb |
|---|---|
| oai_identifier_str |
oai:repositorio.unal.edu.co:unal/77923 |
| network_acronym_str |
UNACIONAL2 |
| network_name_str |
Universidad Nacional de Colombia |
| repository_id_str |
|
| dc.title.spa.fl_str_mv |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| title |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| spellingShingle |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR 660 - Ingeniería química 570 - Biología 664 - Tecnología de alimentos 628 - Ingeniería sanitaria E. coli O157: H7 Absolute quantification Reference material Method validation Digital PCR E. coli O157:H7 PCR digital Cuantificación absoluta Material de referencia Validación de método |
| title_short |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| title_full |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| title_fullStr |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| title_full_unstemmed |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| title_sort |
Desarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCR |
| dc.creator.fl_str_mv |
Tere Peña, Claudia Patricia |
| dc.contributor.advisor.spa.fl_str_mv |
Calderón Ozuna, Martha Nancy Leguizamón Guerrero, Jhon Emerson |
| dc.contributor.author.spa.fl_str_mv |
Tere Peña, Claudia Patricia |
| dc.contributor.corporatename.spa.fl_str_mv |
Universidad Nacional de Colombia Instituto Nacional de Metrología |
| dc.contributor.researchgroup.spa.fl_str_mv |
Bioquímica y Biología Molecular de las Micobacterias |
| dc.subject.ddc.spa.fl_str_mv |
660 - Ingeniería química 570 - Biología 664 - Tecnología de alimentos 628 - Ingeniería sanitaria |
| topic |
660 - Ingeniería química 570 - Biología 664 - Tecnología de alimentos 628 - Ingeniería sanitaria E. coli O157: H7 Absolute quantification Reference material Method validation Digital PCR E. coli O157:H7 PCR digital Cuantificación absoluta Material de referencia Validación de método |
| dc.subject.proposal.eng.fl_str_mv |
E. coli O157: H7 Absolute quantification Reference material Method validation Digital PCR |
| dc.subject.proposal.spa.fl_str_mv |
E. coli O157:H7 PCR digital Cuantificación absoluta Material de referencia Validación de método |
| description |
The E. coli O157: H7 is an enterohemorrhagic bacteria producing Shiga toxin. It can trigger diseases such as hemolytic uremic syndrome (HUS) and hemorrhagic colitis. It has been found associated with outbreaks of foodborne illness. Therefore, it is important to ensure their absence in the production chain of agroindustrial products through the use of sensitive, fast and specific measurement systems such as PCR. There are currently different methods to identify and quantify E. coli O157: H7 in several matrices, but there is no equivalence or comparability between the results of the measurements produced by these methods, due to the absence of traceable reference materials to the international system of units. In order to contribute to the comparability and traceability of the measures carried out by methods based on nucleic acid amplification, a candidate for reference material (MR) of genomic DNA from E. coli O157: H7 was first made in Colombia. After optimizing the culture and DNA extraction conditions of the microorganism, two batches were prepared with concentrations of 172 ± 30 copies / µL and 164864 ± 13096 copies / µL (Uα=0,95; k=2), they proved to be homogeneous and stable for 3 months. In the characterization of the MR candidate a method was used by digital PCR in droplet mode, validated to detect and quantify E. coli O157: H7 DNA in a measurement range of 8 to 8000 copies/μL with measurement uncertainty between 1.5 to 10% depending on the concentration level. The reference material produced could be used for the quality control of the measurements made by real-time PCR, allowing to establish equivalences between the measurement results obtained by the different commercial tests and guarantee traceability to the international system of units. |
| publishDate |
2020 |
| dc.date.accessioned.spa.fl_str_mv |
2020-08-05T02:58:31Z |
| dc.date.available.spa.fl_str_mv |
2020-08-05T02:58:31Z |
| dc.date.issued.spa.fl_str_mv |
2020-02-14 |
| dc.type.spa.fl_str_mv |
Documento de trabajo |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/workingPaper |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/acceptedVersion |
| dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_8042 |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/WP |
| format |
http://purl.org/coar/resource_type/c_8042 |
| status_str |
acceptedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unal.edu.co/handle/unal/77923 |
| url |
https://repositorio.unal.edu.co/handle/unal/77923 |
| dc.language.iso.spa.fl_str_mv |
spa |
| language |
spa |
| dc.relation.references.spa.fl_str_mv |
[3] World health organization, “Foodborne Disease Burden.” [Online]. Available: https://extranet.who.int/sree/Reports?op=vs&path=/WHO_HQ_Reports/G36/PROD/EXT/FoodborneDiseaseBurden. [Accessed: 12-Jan-2020]. [1] Food & Drugs Administration (FDA), “Qué es una enfermedad transmitida por los alimentos.” [Online]. Available: https://www.fda.gov/food/people-risk-foodborne-illness/que-es-una-enfermedad-transmitida-por-los-alimentos. [Accessed: 13-Jan-2020]. [2] Organización Mundial de la Salud (OMS), “Enfermedades de transmisión alimentaria,” 2016. [4] S. E. Majowicz et al., “Global incidence of human Shiga toxin-producing Escherichia coli infections and deaths: a systematic review and knowledge synthesis.,” Foodborne Pathog. Dis., vol. 11, no. 6, pp. 447–55, 2014. [5] W. Gossman, A. Wasey, and P. Salen, Escherichia Coli (E. Coli 0157: H7). 2019. [6] B. Devleesschauwer, S. M. Pires, I. Young, A. Gill, and S. E. Majowicz, “Associating sporadic, foodborne illness caused by Shiga toxin-producing Escherichia coli with specific foods: A systematic review and meta-analysis of case-control studies,” Epidemiol. Infect., vol. 147, 2019. [7] S. Wang et al., “Food safety trends: from globalization of whole genome sequencing to application of new tools to prevent foodborne diseases,” Trends Food Sci. Technol., vol. 57, pp. 188–198, 2016. [8] D. Y. C. Fung, “Rapid methods and automation in microbiology: A review,” Irish J. Agric. Food Res., vol. 39, no. 2, pp. 301–307, 2000. [9] M. Ricchi et al., “Comparison among the quantification of bacterial pathogens by qPCR, dPCR, and cultural methods,” Front. Microbiol., vol. 8, no. JUN, pp. 1–15, 2017. [10] G. A. M. Tarr et al., “Performance of commercial tests for molecular detection of Shiga toxin-producing Escherichia coli (STEC): A systematic review and meta-analysis protocol,” BMJ Open, vol. 9, no. 3, pp. 1–6, 2019. [11] A. Garrido, M.-J. Chapela, B. Román, P. Fajardo, J. M. Vieites, and A. G. Cabado, “In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes,” Int. J. Food Microbiol., vol. 164, no. 1, pp. 92–98, 2013. [12] J. Cloke et al., “Validation of the thermo scientific SureTect Escherichia coli O157:H7 real-time PCR assay for raw beef and produce matrixes,” J. AOAC Int., vol. 98, no. 5, pp. 1301–1314, 2015. [13] I. Son, R. Binet, A. Maounounen-Laasri, A. Lin, T. S. Hammack, and J. A. Kase, “Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR,” Food Microbiol., vol. 40, 2014. [14] B. Li, H. Liu, and W. Wang, “Multiplex real-time PCR assay for detection of Escherichia coli O157 : H7 and screening for non-O157 Shiga toxin-producing E . coli,” pp. 1–13, 2017. [15] V. Barwick and S. Wood, “Achieving metrological traceability in chemical and bioanalytical measurement,” J. Anal. At. Spectrom., vol. 25, no. 6, pp. 785–799, 2010. [16] L. Wang et al., “Development of a Reference Standard of Escherichia coli DNA for Residual DNA Determination in China,” vol. 8, no. 9, pp. 1–6, 2013. [17] P. Van Iwaarden et al., “Certification of a Reference Material of Purified Genomic DNA from Escherichia Coli O157 Certified Reference Material IRMM-449,” 2006. [18] A. S. Devonshire, R. Elaswarapu, and C. A. Foy, “Applicability of RNA standards for evaluating RT-qPCR assays and platforms,” BMC Genomics, vol. 12, p. 10, Feb. 2011. [19] American Type Culture Collection (ATCC), “Certified Reference Materials (CRMs).” [Online]. Available: https://www.atcc.org/en/Standards/Standards_Programs/Certified_Reference_Materials_CRMs.aspx. [Accessed: 13-Jan-2020]. [20] P. Corbisier et al., “CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue,” Metrologia, vol. 49, no. 1A, pp. 08002–08002, 2012. [21] S. Bhat and K. R. Emslie, “Digital polymerase chain reaction for characterisation of DNA reference materials,” Biomol. Detect. Quantif., vol. 10, pp. 47–49, 2016. [22] Proexport, “Programa de transformación productiva,” El priódico las oportunidades, pp. 1–24, 2012. [23] G. G. Moy and Y. Motarjemi, Public Health Measures: International Standards and Harmonization of Food Safety Legislation BT - Encyclopedia of Food Safety, vol. 4. Elsevier Ltd., 2014. [24] INS (Instituto Nacional de Salud), “Protocolo de vigilancia y control de enfermedades transmitidas por alimentos,” Bogotá D.C., 2011. [25] Ministerio de Salud y Protección Social, Resolución 4393 de 1991. Colombia, 1991, p. 3. [26] INVIMA, “Parametros microbiologicos de alimentos.,” 2011. . [27] Ministerio de Seguridad en Alimentos y Fármacos, “Food Code Corea Article 5.” [28] C. U. Europea, Reglamento (CE) n o 2073/2005 de la Comisión, de 15 de noviembre de 2005 , relativo a los criterios microbiológicos aplicables a los productos alimenticios (Texto pertinente a efectos del EEE), vol. 48. 2005, pp. 1–33. [29] INVIMA, “Portafolio de servicios,” 2015. [30] W. H. Organization, “Estimaciones de la OMS sobre la carga mundial de enfermedades de transmisión alimentaria,” World Heal. Organ., vol. 14, p. 2, 2015. [31] Jaime Alberto Guerrero, “Enfermedades Transmitidas por alimentos. PROTOCOLO DE VIGILANCIA EN SALUD PUBLICA,” Inst. Nac. Salud, pp. 3–4, 2016. [32] Instituto Nacional de Salud (INS), BES Semana epidemiológica 52 23 al 29 de Diciembre de 2018. 2018. [33] Organización Mundial de la Salud (OMS), “Riesgos microbiológicos.” [Online]. Available: https://www.who.int/foodsafety/areas_work/microbiological-risks/es/. [Accessed: 13-Jan-2020]. [34] A. H. Havelaar et al., “World Health Organization Global Estimates and Regional Comparisons of the Burden of Foodborne Disease in 2010.” [35] J. B. Kaper, J. P. Nataro, and H. L. T. Mobley, “Pathogenic Escherichia coli,” Nat. Rev. Microbiol., vol. 2, no. 2, pp. 123–140, 2004. [36] FAO, “Escherichia coli,” Bol. enfermedades Transfront. los Anim., p. 39, 2011. [37] Foodborne disease burden epidemiology reference group, WHO estimates of the Global Burden of Foodborne Disease. 2015. [38] Y. Puig Peña, V. Leyva Castillo, N. Apórtela López, N. Campos González, Y. Frerer Marquez, and P. Soto Rodriguez, “Serogrupos y resistencia antimicrobiana de cepas de escherichia coli aisladas en alimentos procedentes de brotes de enfermedades diarreicas,” Rev. Cuba. Aliment. y Nutr., vol. 2, pp. 161–172, 2014. [39] F. Molina, E. López-acedo, R. Tabla, I. Roa, A. Gómez, and J. E. Rebollo, “Improved detection of Escherichia coli and coliform bacteria by multiplex PCR,” ???, pp. 1–9, 2015. [40] J. Y. L. J. W. Y. and C. J. Hovde, “A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157,” J Microbiol Biotechnol., vol. 20, no. 1, pp. 5–14, 2013. [41] C. Farrokh et al., “Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production,” Int. J. Food Microbiol., vol. 162, no. 2, pp. 190–212, 2013. [42] ISO, ISO 16649 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of b-glucuronidase-positive Escherichia coli. 2001. [43] ISO, “ISO 16649-2 Horizontal method for the enumeration of b-glucuronidase-positive Escherichia coli Part 2,” 2001. [44] A. Camacho, M. Giles, A. Ortegón, M. Palao, B. Serrano, and O. Velázquez, “Preparación y dilución de muestras de alimentos para su análisis microbiológico,” p. 9, 2009 [45] N. Da Silva, M. H. Taniwaki, V. C. Junqueira, N. Silveira, M. da Silda do Nascimento, and R. Romeiro Gomes, Microbiological examination methods of food and water : a laboratory manual. 2013. [46] ISO, ISO 16654:2001. Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Escherichia coli 0157, no. 1. 2001. [47] International Organization for Standardization (ISO), “ISO 13136:2012 - Microbiology of food and animal feed — Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens — Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determi.” [Online]. Available: https://www.iso.org/standard/53328.html. [Accessed: 13-Jan-2020]. [48] C. A. Baker, P. M. Rubinelli, S. H. Park, F. Carbonero, and S. C. Ricke, “Shiga toxin-producing Escherichia coli in food: Incidence, ecology, and detection strategies,” Food Control, vol. 59. p. 12, 2016. [49] S. Henson and R. Loader, “Barriers to agricultural exports from developing countries: The role of sanitary and phytosanitary requirements,” World Dev., vol. 29, no. 1, pp. 85–102, 2001. [50] European Comission, “RASFF - Food and Feed Safety Alerts - European Commission.” [Online]. Available: http://ec.europa.eu/food/safety/rasff_en. [Accessed: 25-Apr-2017]. [51] L. E. Forero, “Regulación SPS de Alimentos para exportación a los Estados Unidos,” 2013. [52] European comissión, “RASFF Portal,” 2020. [Online]. Available: https://webgate.ec.europa.eu/rasff-window/portal/?event=searchResultList. [Accessed: 13-Jan-2020]. [53] Ministerio de Salud y protección Social, “Resolucion 3929 de 2013 Requerimientos microbiológicos jugos.” 2013. [54] M. Mangal, S. Bansal, S. K. Sharma, and R. K. Gupta, “Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety,” Crit. Rev. Food Sci. Nutr., vol. 56, no. 9, pp. 1568–1584, Jul. 2016. [55] T. Kuchta et al., “A decade with nucleic acid-based microbiological methods in safety control of foods,” Lett. Appl. Microbiol., vol. 59, no. 3, pp. 263–271, 2014. [56] ISO, “ISO 4833-1 Horizontal method for the enumeration of microorganism,” vol. 44, no. 0, 2013. [57] P. K. Mandal, A. K. Biswas, K. Choi, and U. K. Pal, “Methods for Rapid Detection of Foodborne Pathogens: An Overview,” American Journal of Food Technology, vol. 6, no. 2. pp. 87–102, 2011. [58] G. Bou, A. Fernández-Olmos, C. García, J. A. Sáez-Nieto, and S. Valdezate, “Métodos de identificación bacteriana en el laboratorio de microbiología,” Enferm. Infecc. Microbiol. Clin., vol. 29, no. 8, pp. 601–608, 2011. [59] G. López-Campos, J. V. Martínez-Suárez, M. Aguado-Urda, and V. López-Alonso, “Microarray Detection and Characterization of Bacterial Foodborne Pathogens,” Food, Heal. Nutr., pp. 13–33, 2012. [60] J. W.-F. Law, N.-S. Ab Mutalib, K.-G. Chan, and L.-H. Lee, “Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations.,” Front. Microbiol., vol. 5, no. January, p. 770, 2015. [61] R. L. Bell, K. G. Jarvis, A. R. Ottesen, M. A. Mcfarland, and E. W. Brown, “Recent and emerging innovations in Salmonella detection: A food and environmental perspective,” Microb. Biotechnol., vol. 9, no. 3, pp. 279–292, 2016. [62] S. Bonetta et al., “Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay,” Environ. Sci. Pollut. Res., vol. 23, no. 15, pp. 15302–15309, 2016. [63] N. González-Escalona, E. W. Brown, and G. Zhang, “Development and evaluation of a multiplex real-time PCR (qPCR) assay targeting ttrRSBCA locus and invA gene for accurate detection of Salmonella spp. in fresh produce and eggs,” Food Res. Int., vol. 48, no. 1, pp. 202–208, 2012. [64] A. Rohde et al., “Overview of validated alternative methods for the detection of foodborne bacterial pathogens,” Trends Food Sci. Technol., vol. 62, pp. 113–118, 2017. [65] Z. P. Guan, Y. Jiang, F. Gao, L. Zhang, G. H. Zhou, and Z. J. Guan, “Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR,” Eur. Food Res. Technol., vol. 237, no. 4, pp. 627–637, 2013. [66] M. Varshney, L. Yang, X.-L. Su, and Y. Li, “Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157:H7 in ground beef.,” J. Food Prot., vol. 68, no. 9, pp. 1804–11, 2005. [67] V. Velusamy, K. Arshak, O. Korostynska, K. Oliwa, and C. Adley, “An overview of foodborne pathogen detection: In the perspective of biosensors,” Biotechnol. Adv., vol. 28, no. 2, pp. 232–254, 2010. [68] M. Pohanka, P. Skládal, and O. Pavliš, “Label‐Free Piezoelectric Immunosensor for Rapid Assay of Escherichia coli,” J. Immunoass. Immunochem., vol. 29, no. 1, pp. 70–79, 2007. [69] M. Vaz-Velho, G. Duarte, and P. Gibbs, “Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish,” J. Microbiol. Methods, vol. 40, no. 2, pp. 147–151, 2000. [70] D. Emerson, L. Agulto, H. Liu, and L. Liu, “Identifying and Characterizing Bacteria in an Era of Genomics and Proteomics,” Bioscience, vol. 58, no. 10, p. 925, 2008. [71] T. C. Chiu, “Recent advances in bacteria identification by matrix-assisted laser desorption/ionization mass spectrometry using nanomaterials as affinity probes,” Int. J. Mol. Sci., vol. 15, no. 5, pp. 7266–7280, 2014. [72] P.-E. Fournier, M. Drancourt, P. Colson, J.-M. Rolain, B. La Scola, and D. Raoult, “Modern clinical microbiology: new challenges and solutions,” Nat. Rev. Microbiol., vol. 11, no. 8, pp. 574–585, 2013. [73] N. T. Salihah, M. M. Hossain, H. Lubis, and M. U. Ahmed, “Trends and advances in food analysis by real-time polymerase chain reaction,” J. Food Sci. Technol., vol. 53, no. 5, pp. 2196–2209, 2016. [74] INVIMA, “Portafolio de servicios INVIMA,” pp. 1–7, 2018. [75] T. González Flores and R. A. Rojas Herrera, “Enfermedades transmitidas por alimentos y PCR: Prevención y diagnóstico,” Salud Publica Mex., vol. 47, no. 5, pp. 388–390, 2005. [76] M. T. Rahman, M. S. Uddin, R. Sultana, A. Moue, and M. Setu, “Polymerase Chain Reaction (PCR): A Short Review,” Anwer Khan Mod. Med. Coll. J., vol. 4, no. 1, pp. 30–36, 2013. [77] H. Ringuet et al., “hsp65 Sequencing for identification of rapidly growing mycobacteria,” J. Clin. Microbiol., vol. 37, no. 3, pp. 852–857, 1999. [78] G. W. Payne, P. Vandamme, S. H. Morgan, J. J. Lipuma, and T. Coenye, “Development of a recA gene-based identification approach for the entire Burkholderia genus,” Appl. Environ. Microbiol., vol. 71, no. 7, pp. 3917–3927, 2005. [79] L. Dong, Y. Meng, Z. Sui, J. Wang, L. Wu, and B. Fu, “Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.,” Sci. Rep., vol. 5, 2015. [80] S. Dhanasekaran, T. M. Doherty, and J. Kenneth, “Comparison of different standards for real-time PCR-based absolute quantification,” J. Immunol. Methods, vol. 354, no. 1–2, pp. 34–39, 2010. [81] S. Bhat and K. R. Emslie, “Digital polymerase chain reaction for characterization of DNA reference materials,” Biomol. Detect. Quantif., pp. 3–5, 2016. [82] L. Tamay de Dios, C. Ibarra, and C. Velasquillo, “Fundamentos de la reacción en cadena de la polimerasa (PCR) y de la PCR en tiempo real,” Learn. Discip. ICLS 2010 Conf. Proc. - 9th Int. Conf. Learn. Sci., vol. 12, pp. 70–78, 2013. [83] M. Arya, I. S. Shergill, M. Williamson, L. Gommersall, N. Arya, and H. R. H. Patel, “Basic principles of real-time quantitative PCR,” Expert Rev. Mol. Diagn., vol. 5, no. 2, pp. 209–219, 2005. [84] Y. Li, X. Zhou, and D. Ye, “Molecular beacons: An optimal multifunctional biological probe,” Biochem. Biophys. Res. Commun., vol. 373, no. 4, pp. 457–461, 2008. [85] E. Omiccioli, G. Amagliani, G. Brandi, and M. Magnani, “A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk,” Food Microbiol., vol. 26, no. 6, pp. 615–622, 2009. [86] S. H. Liming and A. A. Bhagwat, “Application of a molecular beacon - Real-time PCR technology to detect Salmonella species contaminating fruits and vegetables,” Int. J. Food Microbiol., vol. 95, no. 2, pp. 177–187, 2004. [87] S. Perelle, F. Dilasser, J. Grout, and P. Fach, “Detection by 5′-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world’s most frequent clinical cases,” Mol. Cell. Probes, vol. 18, no. 3, pp. 185–192, 2004. [88] L. W. Noll et al., “A Four-Plex Real-Time PCR Assay, Based on rfb E, stx 1, stx 2, and eae Genes, for the Detection and Quantification of Shiga Toxin–Producing Escherichia coli O157 in Cattle Feces,” Foodborne Pathog. Dis., vol. 12, no. 9, pp. 787–794, 2015. [89] G. A. Leotta et al., “Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga,” Rev. Argent. Microbiol., vol. 37, no. 1, pp. 1–10, 2005. [90] B. Vogelstein and K. W. Kinzler, “Digital PCR,” Genetics, vol. 96, no. August, p. 92369241, 1999. [91] A. A. Morley, “Digital PCR: A brief history,” Biomol. Detect. Quantif., vol. 1, no. 1, pp. 1–2, 2014. [92] J. F. Huggett, J. A. Garson, and A. S. Whale, “Digital PCR and Its Potential Application to Microbiology,” Mol. Microbiol. Diagnostic Princ. ans Pract., pp. 49–57, 2016. [93] M. Wang et al., “Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk,” Int. J. Food Microbiol., vol. 266, 2018. [94] J. F. Huggett, S. Cowen, and C. A. Foy, “Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool,” vol. 1, no. 61, pp. 79–88, 2015. [95] B. Verhaegen, K. De Reu, L. De Zutter, K. Verstraete, M. Heyndrickx, and E. Van Coillie, “Comparison of droplet digital PCR and qPCR for the quantification of shiga toxin-producing Escherichia coli in bovine feces,” Toxins (Basel)., vol. 8, no. 5, pp. 1–11, 2016. [96] R. Sanders, D. J. Mason, C. A. Foy, and J. F. Huggett, “Evaluation of Digital PCR for Absolute RNA Quantification,” PLoS One, vol. 8, no. 9, pp. 1–9, 2013. [97] D. G. Burke et al., “Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity,” Anal. Chem., vol. 85, no. 3, pp. 1657–1664, 2013. [98] R. Sanders, J. F. Huggett, C. A. Bushell, S. Cowen, D. J. Scott, and C. A. Foy, “Evaluation of digital PCR for absolute DNA quantification,” Anal. Chem., vol. 83, no. 17, pp. 6474–6484, 2011. [99] S. Bhat, N. Curach, T. Mostyn, G. S. Bains, K. R. Griffiths, and K. R. Emslie, “Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units,” Anal. Chem., vol. 82, no. 17, pp. 7185–7192, 2010. [100] Biomerieux, “Métodos Rápidos para el Métodos Rápidos para el control Microbiológico de Alimentos,” pp. 1–54, 2011. [101] G. Pohl and M. Shih Ie, “Principle and applications of digital PCR,” Expert Rev Mol Diagn, vol. 4, no. 1, pp. 41–47, 2004. [102] M. Boyer and J. Combrisson, “Analytical opportunities of quantitative polymerase chain reaction in dairy microbiology,” Int. Dairy J., vol. 30, no. 1, pp. 45–52, 2013. [103] G. Agustí, M. Fittipaldi, and F. Codony, “Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples,” Curr. Microbiol., vol. 75, no. 6, pp. 779–785, 2018. [104] S. Broeders et al., “Guidelines for validation of qualitative real-time PCR methods,” Trends Food Sci. Technol., vol. 37, no. 2, pp. 115–126, 2014. [105] J. F. Huggett, S. Cowen, and C. A. Foy, “Considerations for digital PCR as an accurate molecular diagnostic tool,” Clin. Chem., vol. 61, no. 1, pp. 79–88, 2015. [106] P. Truchado, M. I. Gil, T. Kostic, and A. Allende, “Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production,” Food Control, vol. 62, pp. 150–156, 2016. [107] E. T. Gensberg, M. Polt, M. Konrad-k, P. Kinner, A. Sessitsch, and T. Kostic, “Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality,” Water Res., vol. 67, no. 0, pp. 367–376, 2014. [108] B. Li, Z. Hu, and C. a Elkins, “Detection of live Escherichia coli O157:H7 cells by PMA-qPCR.,” J. Vis. Exp., no. 84, p. e50967, 2014. [109] M. Chapela, A. Garrido-Maestu, and A. G. Cabado, “Detection of foodborne pathogens by qPCR : A practical approach for food industry applications,” Cogent Food Agric., vol. 1, no. 1, pp. 1–19, 2015. [110] J. Pavšič et al., “Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA,” Anal. Bioanal. Chem., 2017. [111] H. J. He, J. L. Almeida, S. P. Lund, C. R. Steffen, S. Choquette, and K. D. Cole, “Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements,” Biomol. Detect. Quantif., vol. 8, pp. 1–8, 2016. [112] C. Floren, I. Wiedemann, B. Brenig, E. Schütz, and J. Beck, “Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR),” Food Chem., vol. 173, pp. 1054–1058, 2015. [113] M. Milavec, K. Gruden, and Z. Jana, “Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR,” PLoS One, vol. 8, no. 5, 2013. [114] H. White et al., “A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR,” Leukemia, vol. 29, no. 2, pp. 369–376, 2015. [115] C. M. Hindson et al., “Absolute quantification by droplet digital PCR versus analog real-time PCR,” Nat. Methods, vol. 10, no. 10, pp. 1003–1005, 2013. [116] M. C. Kline, E. L. Romsos, and D. L. Duewer, “Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets,” Anal. Chem., p. acs.analchem.5b03692, 2016. [117] J. Pavšič, J. Žel, and M. Milavec, “Digital PCR for direct quantification of viruses without DNA extraction,” Anal. Bioanal. Chem., vol. 408, no. 1, pp. 67–75, 2016. [118] L. Gerdes, A. Iwobi, U. Busch, and S. Pecoraro, “Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms,” Biomol. Detect. Quantif., vol. 7, pp. 9–20, 2016. [119] M. Sivaganesan, M. Varma, S. Siefring, and R. Haugland, “Quantification of plasmid DNA standards for U.S. EPA fecal indicator bacteria qPCR methods by droplet digital PCR analysis,” J. Microbiol. Methods, vol. 152, pp. 135–142, Sep. 2018. [120] D. L. Duewer, M. C. Kline, E. L. Romsos, and B. Toman, “Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter,” Anal. Bioanal. Chem., vol. 410, no. 12, 2018. [121] J. F. Huggett et al., “The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.,” Clin. Chem., vol. 59, no. 6, pp. 892–902, Jun. 2013. [122] T. Soejima, J. I. Minami, T. Yaeshima, and K. Iwatsuki, “An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk,” Appl. Microbiol. Biotechnol., vol. 95, no. 2, pp. 485–497, 2012. [123] A. Rompré, P. Servais, J. Baudart, M. R. De-Roubin, and P. Laurent, “Detection and enumeration of coliforms in drinking water: Current methods and emerging approaches,” J. Microbiol. Methods, vol. 49, no. 1, pp. 31–54, 2002. [124] Adria Developpement, “Validation of alternative analytical methods Application in food microbiology Summary report EN ISO 16140 validation study of the DuPont TM BAX detection of Salmonella spp in meat products , egg,” 2015. [125] M. L. Camaró-Sala, R. Martínez-García, P. Olmos-Martínez, V. Catalá-Cuenca, M. D. Ocete-Mochón, and C. Gimeno-Cardona, “Validación y verificación analítica de los métodos microbiológicos,” Enfermedades Infecc. y Microbiol. Clin. Monogr., vol. 33, no. 7, pp. e31–e36, 2015. [126] AFNOR, “E.coli O157 methods.” [Online]. Available: https://nf-validation.afnor.org/en/food-industry/e-coli-o157/. [Accessed: 14-Jan-2020]. [127] AOAC, “Official Methods of Analysis.” [Online]. Available: http://www.eoma.aoac.org/. [Accessed: 14-Jan-2020]. [128] AFNOR, “Validation of alternative analytical methods Application in food microbiology Molecular Detection Assay E. coli O157 (including H7) for the detection of Escherichia coli O157 (including H7) in raw beef meat, raw dairy products, raw fruits & vegetables,” 2016. [129] ISHA, “NF VALIDATION 16140 TM AFNOR CERTIFICATION VALIDATION OF THE METHOD ADIAFOOD Escherichia coli O157 : H7 method ( single well ) For the detection of Escherichia coli O157 : H7 SUMMARY REPORT ‐ SEPTEMBER 2015 – V1 Expert laboratory : ISHA 25 avenue de la Ré,” 2015. [130] Adria Developpement, “Rapport de synthèse Reconduction de la validation EN ISO 16140 de la méthode Bax® E. coli O157:H7 MP,” 2012. [131] Adria Developpement, “Validation study according to the EN ISO 16140 standard Summary report EN ISO 16140 validation of the DuPont TM BAX ® Real-Time PCR Assay for E . coli O157 : H7 method in raw beef meats and raw vegetables,” 2014. [132] AFNOR, “Validation of alternative analytical methods Application in food microbiology validation study of the GeneDisc® method for the simultaneous detection of Salmonella spp. and Escherichia coli O157:H7-Detection of Escherichia coli O157:H7,” 2017. [133] Adria Developpement, “Rapport de synthèse Validation EN ISO 16140 de la méthode Méthode iQ-Check Eschericia coli O157:H7,” 2012. [134] Adria Developpement, “Summary report EN ISO 16140 validation study of the MicroSEQ ® Escherichia coli O157 : H7 method for the detection of Escherichia coli O157 : H7 in raw beef meats and raw vegetables,” 2015. [135] S. Pillet et al., “Comparative Evaluation of Six Commercialized Multiplex PCR Kits for the Diagnosis of Respiratory Infections,” PLoS One, vol. 8, no. 8, 2013. [136] P.-Y. Cheung, C. W. Chan, W. Wong, T. L. Cheung, and K. M. Kam, “Evaluation of two real-time polymerase chain reaction pathogen detection kits for Salmonella spp. in food,” Lett. Appl. Microbiol., vol. 39, no. 6, pp. 509–515, Dec. 2004. [137] EURACHEM, “La Adecuación al Uso de los Métodos Analíticos.” 2016, p. 66, 2016. [138] JCGM (Comité Conjunto para las Guías en Metrología), Vocabulario Internacional de Metrología. 2008. [139] International Organization for Standardization, ISO 17511 INvitro diagnostic medical devices-Measurement of quantities in biological samples - Metrological traceability of values assigned to calibrators and control materials, vol. 2005. 2006. [140] M. Thompson, S. L. R. Ellison, and R. Wood, “Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC Technical Report),” Int. Union Pure Appl. Chem., vol. 74, no. 5, pp. 835–855, 2002. [141] S. L. R. Ellison, M. Rosslein, A. Williams, L. A. Konopelko, and A. V. Garmash, “EURACHEM/CITAC Guide: Quantifying Uncertainty in Analytical Measurement,” Journal of Analytical Chemistry, vol. 58, no. 2. European Federation of National Associations of Analytical Laboratories, p. 191, 2003. [142] BIPM, International vocabulary of metrology - Basic and general concepts and associated terms (VIM). 2012. [143] EURACHEM and B. King, “The selection and use of reference materials,” 2002. [144] “ISO/Guide 33:2015(en), Reference materials — Good practice in using reference materials.” [Online]. Available: https://www.iso.org/obp/ui/#iso:std:iso:guide:33:ed-3:v1:en. [Accessed: 14-Jan-2020]. [145] W. R. Wolf, “History of reference materials for food and nutrition metrology: As represented in the series of BERM symposia,” Anal. Bioanal. Chem., vol. 397, no. 2, pp. 413–421, 2010. [146] Societa Italiana di Fisica, Metrology: from Physics Fundamentals to Quality of Life. 2017. [147] NIST, “Standard Reference Materials.” [Online]. Available: https://www.nist.gov/srm. [Accessed: 14-Jan-2020]. [148] “Certified Reference Materials catalogue of the JRC.” [Online]. Available: https://crm.jrc.ec.europa.eu/. [Accessed: 14-Jan-2020]. [149] WHO (World Health Organization), “WHO | International reference materials.” [Online]. Available: https://www.who.int/bloodproducts/ref_materials/en/. [Accessed: 14-Jan-2020]. [150] BIPM, “Database of higher-order reference materials, measurement methods/procedures and services.” [Online]. Available: https://www.bipm.org/jctlm/. [Accessed: 13-Jan-2020]. [151] L. Deprez et al., “Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material,” Biomol. Detect. Quantif., vol. 9, pp. 29–39, 2016. [152] P. Van Iwaarden et al., “Certification of a Reference Material of Purified Genomic DNA from Escherichia Coli O157 Certified Reference Material IRMM-449,” vol. 157, no. Edl 933. [153] J. R. C. JRC, Certification of Reference Materials of Maize Seed Powder containing Genetically Modified MON 810 Maize Certified Reference Materials ERM ® -BF413k. . [154] P. Corbisier, “CERTIFICATION REPORT A set of three plasmid DNA calibration solutions bearing a porcine- specific DNA fragment A set of three plasmid DNA calibration solutions bearing a porcine-specific DNA fragment,” 2015. [155] P. Corbisier et al., “DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials,” Anal. Bioanal. Chem., vol. 407, no. 7, pp. 1831–1840, 2015. [156] Zeptometrix Corporation, “Escherichia coli O157:H7; EDL933, Genomic DNA,” 2014, p. 801622. [157] International Organization for Standardization (ISO), “ISO/Guide 30:2015(en), Reference materials — Selected terms and definitions.” [Online]. Available: https://www.iso.org/obp/ui/#iso:std:iso:guide:30:ed-3:v1:en. [Accessed: 14-Jan-2020]. [158] W. A. Schmid and R. J. Lazos Martínez, “Guide to estimate the measurement uncertainty,” Natl. Cent. Metrol. Mex., p. 27, 2000. [159] T. P. J. Linsinger, J. Pauwels, A. Lamberty, H. G. Schimmel, A. M. H. Van Der Veen, and L. Siekmann, “Estimating the uncertainty of stability for matrix CRMs,” Anal. Bioanal. Chem., vol. 370, no. 2–3, pp. 183–188, 2001. [160] A. M. H. Van Der Veen, T. P. J. Linsinger, H. Schimmel, A. Lamberty, and J. Pauwels, “Uncertainty calculations in the certification of reference materials 4. Characterisation and certification,” Accredit. Qual. Assur., vol. 6, no. 7, pp. 290–294, 2001. [161] I. O. for S. ISO, “ISO 17034:2016(es), Requisitos generales para la competencia de los productores de materiales de referencia.” [Online]. Available: https://www.iso.org/obp/ui#iso:std:iso:17034:ed-1:v1:es. [Accessed: 14-Jan-2020]. [162] World Health Organization, “Recommendations for the preparation, characterization and establishment of international and other biological reference standards,” WHO Tech. Rep. Ser. 932, vol. 2, no. 932, pp. 73–131, 2006. [163] International Organization for Standardization (ISO), “ISO - ISO Guide 35:2017 - Reference materials — Guidance for characterization and assessment of homogeneity and stability.” [Online]. Available: https://www.iso.org/standard/60281.html. [Accessed: 13-Jan-2020]. [164] E. D. F. Guimarães, E. C. P. Do Rego, H. C. M. Cunha, J. M. Rodrigues, J. D. F. Villar, and V. S. Da Cunha, “Homogeneity study for certification of a candidate reference material for polycyclic aromatic hydrocarbons,” 19th IMEKO World Congr. 2009, vol. 4, pp. 2377–2381, 2009. [165] T. P. J. Linsinger, J. Pauwels, A. M. H. Van Der Veen, H. Schimmel, and A. Lamberty, “Homogeneity and stability of reference materials,” Accredit. Qual. Assur., vol. 6, no. 1, pp. 20–25, Jan. 2001. [166] T. P. J. Linsinger, J. Pauwels, A. M. H. Van Der Veen, H. Schimmel, and A. Lamberty, “Homogeneity and stability of reference materials,” Accredit. Qual. Assur., vol. 6, no. 1, pp. 20–25, 2001. [167] A. Lamberty, H. Schimmel, and J. Pauwels, “The study of the stability of reference materials by isochronous measurements,” in Fresenius’ Journal of Analytical Chemistry, 1998, vol. 360, no. 3–4, pp. 359–361. [168] ISO Guide 35, “Reference materials -- General and statistical principles for certification.” International Organization for Standardization. Geneva, Suiza, 2006. [169] A. B. Košir et al., “Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR,” Anal. Bioanal. Chem., vol. 409, no. 28, pp. 6689–6697, 2017. [170] J. F. Huggett et al., “The digital MIQE guidelines: Minimum information for publication of quantitative digital PCR experiments,” Clin. Chem., vol. 59, no. 6, pp. 892–902, 2013. [171] K. Andersen and J. Merry, “Reference Materials,” vol. 18, no. 6, pp. 376–383, 1999. [172] E. Theres, M. Polt, M. Konrad-k, P. Kinner, A. Sessitsch, and T. Kosti, “Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality,” vol. 7, no. 0, pp. 367–376, 2014. [173] E. M. Nielsen and M. T. Andersen, “Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5 ′ Nuclease PCR Assay Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5Ј Nuclease PCR Assay,” J. Clin. Microbiol., vol. 41, no. 7, pp. 2884–2893, 2003. [174] I. O. for S. ISO, “ISO/TS 13136:2012 - Microbiology of food and animal feed -- Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens -- Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups,” 2012. [Online]. Available: https://www.iso.org/standard/53328.html. [Accessed: 21-Mar-2019]. [175] Integrated DNA technologies, “How do I use the OligoAnalyzer tool to analyze possible hairpins and dimers formed by my oligo?” [Online]. Available: https://www.idtdna.com/pages/support/faqs/how-do-i-use-the-oligoanalyzer-tool-to-analyze-possible-hairpins-and-dimers-formed-by-my-oligo. [Accessed: 24-Sep-2019]. [176] S. Das Mitrai et al., “Duplex PCR for specific detection of Escherichia coli and its differentiation from other Enterobacteriaceae,” Indian J. Anim. Sci., vol. 85, no. 8, pp. 16–19, 2015. [177] K. Horakova, H. Mlejnkova, and P. Mlejnek, “Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: Cytochrome bd complex, lactose permease, ??-d-glucuronidase, and ??-d-galactosidase,” J. Appl. Microbiol., vol. 105, no. 4, pp. 970–976, 2008. [178] B. Li and J. Chen, “Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157 : H7 Cells by Targeting Z3276 as a Genetic,” vol. 78, no. 15, pp. 5297–5304, 2012. [179] E. M. Nielsen and M. T. Andersen, “Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5 ′ Nuclease PCR Assay Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5Ј Nuclease PCR Assay,” J. Clin. Microbiol., vol. 41, no. 7, pp. 2884–2893, 2003. [180] E. Barbau-Piednoir, S. Bertrand, J. Mahillon, N. H. Roosens, and N. Botteldoorn, “SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels,” Appl. Microbiol. Biotechnol., vol. 97, no. 22, pp. 9811–9824, 2013. [181] BIORAD, “Real-time PCR: Applications Guide,” Bio-Rad Lab., pp. 2–84, 2006. [182] V. K. Singh, R. Govindarajan, S. Naik, and A. Kumar, “The effect of hairpin structure on PCR amplification efficiency.,” Mol Biol Today, vol. 1, no. 3, pp. 67–9, 2000. [183] A. Ud-Din and S. Wahid, “Relationship among Shigella spp. And enteroinvasive Escherichia coli (EIEC) and their differentiation,” Brazilian J. Microbiol., vol. 45, no. 4, pp. 1131–1138, 2014. [184] Bio-Rad, “Droplet Digital TM PCR Applications guide.” [185] Promega, “Assembly of Restriction Enzyme Digestions,” 2007. [186] W. Liang et al., “Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR,” Chem. Cent. J., vol. 10, no. 1, p. 55, 2016. [187] R. T. Hayden et al., “Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus,” J. Clin. Microbiol., vol. 51, no. 2, pp. 540–546, 2013. [188] M. E. Hunter, R. M. Dorazio, J. S. S. Butterfield, G. Meigs-Friend, L. G. Nico, and J. A. Ferrante, “Detection limits of quantitative and digital PCR assays and their influence in presence???absence surveys of environmental DNA,” Mol. Ecol. Resour., vol. 17, no. 2, pp. 221–229, 2017. [189] Thermo Scientific, “T042-TECHNICAL BULLETIN NanoDrop Spectrophotometers.” [190] C. Villamil, “Desarrollo de herramientas de aseguramiento metrológico para la identificación y cuantificación de Salmonella spp. por métodos basados en PCR,” Universidad Nacional de Colombia, 2019. [191] “ISO/Guide 31:2015(en), Reference materials — Contents of certificates, labels and accompanying documentation.” [Online]. Available: https://www.iso.org/obp/ui/#iso:std:iso:guide:31:ed-3:v1:en. [Accessed: 14-Jan-2020]. [192] G. M.M., E. P., L. V., L. M. M., S. L. L., and A. M. L., “[Detection of diarrheagenic Escherichia coli in children from poor neighborhoods in Corrientes, Argentina],” Rev. Cubana Med. Trop., vol. 62, no. 1, pp. 42–47, 2010. [193] A. Paton and J. Paton, “Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex Enterohemorrhagic E . coli hlyA , rfb O111 , and Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx 1 , stx 2 , eae,” J. Clin. Microbiol., vol. 36, no. 2, pp. 598–602, 1998. [194] B. China, V. Pirson, and J. Mainil, “Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes,” Appl. Environ. Microbiol., vol. 62, no. 9, pp. 3462–3465, 1996. [195] W. Liang et al., “Quantification of plasmid DNA reference materials for Shiga toxin ‑ producing Escherichia coli based on UV , HR ‑ ICP ‑ MS and digital PCR,” pp. 1–10, 2016. [196] D. R. Pollard, W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee, “Erratum: Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction (J. Clin. Microbiol., Volume 28, No. 3, P. 542),” J. Clin. Microbiol., vol. 28, no. 6, p. 1491, 1990. [197] Y. Hu, Q. Zhang, and J. C. Meitzler, “Rapid and sensitive detection of Escherichia coli O157 : H7 in bovine faeces by a multiplex PCR,” J. Appl. Microbiol., vol. 87, pp. 867–876, 1999. [198] V. Brusa, L. Galli, L. H. Linares, E. E. Ortega, J. P. Lirón, and G. A. Leotta, “Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat,” J. Microbiol. Methods, vol. 119, pp. 10–17, 2015. [199] K. A. Ziebell, S. C. Read, R. P. Johnson, and C. L. Gyles, “Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins,” Res. Microbiol., vol. 153, no. 5, pp. 289–300, 2002. [200] R. Gordillo, A. Rodríguez, M. L. Werning, E. Bermúdez, and M. Rodríguez, “Quantification of viable Escherichia coli O157:H7 in meat products by duplex real-time PCR assays.,” Meat Sci., vol. 96, no. 2, pp. 964–970, 2014. [201] E. C. Chern, S. Siefring, J. Paar, M. Doolittle, and R. A. Haugland, “Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes,” Lett. Appl. Microbiol., vol. 52, no. 3, pp. 298–306, 2011. [202] E. Frahm and U. Obst, “Application of the fluorogenic probe technique ( TaqMan PCR ) to the detection of Enterococcus spp . and Escherichia coli in water samples,” vol. 52, pp. 123–131, 2003. [203] J. N. Miller and J. C. Miller, Estadística y Quimiometría para Química Analítica, 4a edición. 2002. |
| dc.rights.spa.fl_str_mv |
Derechos reservados - Universidad Nacional de Colombia |
| dc.rights.coar.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| dc.rights.license.spa.fl_str_mv |
Atribución-NoComercial-SinDerivadas 4.0 Internacional |
| dc.rights.spa.spa.fl_str_mv |
Acceso abierto |
| dc.rights.uri.spa.fl_str_mv |
http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| dc.rights.accessrights.spa.fl_str_mv |
info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
Atribución-NoComercial-SinDerivadas 4.0 Internacional Derechos reservados - Universidad Nacional de Colombia Acceso abierto http://creativecommons.org/licenses/by-nc-nd/4.0/ http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.format.extent.spa.fl_str_mv |
159 |
| dc.format.mimetype.spa.fl_str_mv |
application/pdf |
| dc.publisher.program.spa.fl_str_mv |
Bogotá - Ciencias - Maestría en Ciencias - Bioquímica |
| dc.publisher.department.spa.fl_str_mv |
Departamento de Química |
| dc.publisher.branch.spa.fl_str_mv |
Universidad Nacional de Colombia - Sede Bogotá |
| institution |
Universidad Nacional de Colombia |
| bitstream.url.fl_str_mv |
https://repositorio.unal.edu.co/bitstream/unal/77923/1/1020752137_2020.pdf https://repositorio.unal.edu.co/bitstream/unal/77923/2/license.txt https://repositorio.unal.edu.co/bitstream/unal/77923/3/license_rdf https://repositorio.unal.edu.co/bitstream/unal/77923/4/1020752137_2020.pdf.jpg |
| bitstream.checksum.fl_str_mv |
3f3ea0e0fb14ae39145a65346298e7f9 6f3f13b02594d02ad110b3ad534cd5df 217700a34da79ed616c2feb68d4c5e06 2382d0345f56f7ca44351475541664df |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio Institucional Universidad Nacional de Colombia |
| repository.mail.fl_str_mv |
repositorio_nal@unal.edu.co |
| _version_ |
1814090047739133952 |
| spelling |
Atribución-NoComercial-SinDerivadas 4.0 InternacionalDerechos reservados - Universidad Nacional de ColombiaAcceso abiertohttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Calderón Ozuna, Martha Nancy8b96f3c7-a02a-400b-ace4-c710e8a97d83-1Leguizamón Guerrero, Jhon Emersone850977b-a4be-4948-8d68-2a49997fe0d4-1Tere Peña, Claudia Patriciaad5a12c0-137e-481a-bbe6-67e53598a060Universidad Nacional de ColombiaInstituto Nacional de MetrologíaBioquímica y Biología Molecular de las Micobacterias2020-08-05T02:58:31Z2020-08-05T02:58:31Z2020-02-14https://repositorio.unal.edu.co/handle/unal/77923The E. coli O157: H7 is an enterohemorrhagic bacteria producing Shiga toxin. It can trigger diseases such as hemolytic uremic syndrome (HUS) and hemorrhagic colitis. It has been found associated with outbreaks of foodborne illness. Therefore, it is important to ensure their absence in the production chain of agroindustrial products through the use of sensitive, fast and specific measurement systems such as PCR. There are currently different methods to identify and quantify E. coli O157: H7 in several matrices, but there is no equivalence or comparability between the results of the measurements produced by these methods, due to the absence of traceable reference materials to the international system of units. In order to contribute to the comparability and traceability of the measures carried out by methods based on nucleic acid amplification, a candidate for reference material (MR) of genomic DNA from E. coli O157: H7 was first made in Colombia. After optimizing the culture and DNA extraction conditions of the microorganism, two batches were prepared with concentrations of 172 ± 30 copies / µL and 164864 ± 13096 copies / µL (Uα=0,95; k=2), they proved to be homogeneous and stable for 3 months. In the characterization of the MR candidate a method was used by digital PCR in droplet mode, validated to detect and quantify E. coli O157: H7 DNA in a measurement range of 8 to 8000 copies/μL with measurement uncertainty between 1.5 to 10% depending on the concentration level. The reference material produced could be used for the quality control of the measurements made by real-time PCR, allowing to establish equivalences between the measurement results obtained by the different commercial tests and guarantee traceability to the international system of units.E. coli O157:H7 es una bacteria enterohemorrágica productora de toxinas Shiga capaz de desencadenar enfermedades como el síndrome urémico hemolítico (SUH) y colitis hemorrágica. Se ha encontrado asociada a brotes de enfermedades transmitidas por alimentos, por lo que es importante garantizar su ausencia en toda la cadena productiva de los productos agroindustriales a través del uso de sistemas de medición sensibles, rápidos y específicos como la PCR. Actualmente hay diferentes métodos para identificar y cuantificar E. coli O157:H7 en varias matrices, pero no existe equivalencia o comparabilidad entre los resultados de las mediciones producidas por estas metodologías, debido a la ausencia de materiales de referencia trazables al sistema internacional de unidades. Con el objetivo de contribuir a la comparabilidad y trazabilidad de las medidas realizadas por métodos basados en la amplificación de ácidos nucleicos, se produjo en Colombia por primera vez un candidato a material de referencia (MR) de ADN genómico de E. coli O157:H7. Luego de optimizar las condiciones de cultivo y extracción de ADN del microorganismo se prepararon dos lotes con concentraciones de 172 ± 30 copias/µL y 164864 ± 13096 copias/µL (Uα=0,95; k=2), los que demostraron ser homogéneos y estables durante 3 meses. En la caracterización del candidato a MR se empleó un método por PCR digital en modo gota, previamente validado para detectar y cuantificar ADN de E. coli O157:H7 en un intervalo de medición de 8 a 8000 copias/μL con incertidumbre de medición entre 1.5 a 10% según el nivel de concentración. El material de referencia producido se podría emplear para el control de calidad de las medidas realizadas por PCR tiempo real, permitiendo establecer equivalencias entre los resultados de medición obtenidos por los distintos ensayos comerciales y garantizar la trazabilidad al sistema internacional de unidades.Ministerio de CienciasDesarrollo de materiales de referencia o biometrológicos para la detección de Escherichia coli y Salmonella spp. en productos agroindustriales del programa de transformación productivaLínea de Investigación: BioprocesosMaestría159application/pdfspa660 - Ingeniería química570 - Biología664 - Tecnología de alimentos628 - Ingeniería sanitariaE. coli O157: H7Absolute quantificationReference materialMethod validationDigital PCRE. coli O157:H7PCR digitalCuantificación absolutaMaterial de referenciaValidación de métodoDesarrollo de un candidato a material de referencia para la detección y cuantificación de Escherichia coli O157 H7 por PCRDocumento de trabajoinfo:eu-repo/semantics/workingPaperinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_8042Texthttp://purl.org/redcol/resource_type/WPBogotá - Ciencias - Maestría en Ciencias - BioquímicaDepartamento de QuímicaUniversidad Nacional de Colombia - Sede Bogotá[3] World health organization, “Foodborne Disease Burden.” [Online]. Available: https://extranet.who.int/sree/Reports?op=vs&path=/WHO_HQ_Reports/G36/PROD/EXT/FoodborneDiseaseBurden. [Accessed: 12-Jan-2020].[1] Food & Drugs Administration (FDA), “Qué es una enfermedad transmitida por los alimentos.” [Online]. Available: https://www.fda.gov/food/people-risk-foodborne-illness/que-es-una-enfermedad-transmitida-por-los-alimentos. [Accessed: 13-Jan-2020].[2] Organización Mundial de la Salud (OMS), “Enfermedades de transmisión alimentaria,” 2016.[4] S. E. Majowicz et al., “Global incidence of human Shiga toxin-producing Escherichia coli infections and deaths: a systematic review and knowledge synthesis.,” Foodborne Pathog. Dis., vol. 11, no. 6, pp. 447–55, 2014.[5] W. Gossman, A. Wasey, and P. Salen, Escherichia Coli (E. Coli 0157: H7). 2019.[6] B. Devleesschauwer, S. M. Pires, I. Young, A. Gill, and S. E. Majowicz, “Associating sporadic, foodborne illness caused by Shiga toxin-producing Escherichia coli with specific foods: A systematic review and meta-analysis of case-control studies,” Epidemiol. Infect., vol. 147, 2019.[7] S. Wang et al., “Food safety trends: from globalization of whole genome sequencing to application of new tools to prevent foodborne diseases,” Trends Food Sci. Technol., vol. 57, pp. 188–198, 2016.[8] D. Y. C. Fung, “Rapid methods and automation in microbiology: A review,” Irish J. Agric. Food Res., vol. 39, no. 2, pp. 301–307, 2000.[9] M. Ricchi et al., “Comparison among the quantification of bacterial pathogens by qPCR, dPCR, and cultural methods,” Front. Microbiol., vol. 8, no. JUN, pp. 1–15, 2017.[10] G. A. M. Tarr et al., “Performance of commercial tests for molecular detection of Shiga toxin-producing Escherichia coli (STEC): A systematic review and meta-analysis protocol,” BMJ Open, vol. 9, no. 3, pp. 1–6, 2019.[11] A. Garrido, M.-J. Chapela, B. Román, P. Fajardo, J. M. Vieites, and A. G. Cabado, “In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes,” Int. J. Food Microbiol., vol. 164, no. 1, pp. 92–98, 2013.[12] J. Cloke et al., “Validation of the thermo scientific SureTect Escherichia coli O157:H7 real-time PCR assay for raw beef and produce matrixes,” J. AOAC Int., vol. 98, no. 5, pp. 1301–1314, 2015.[13] I. Son, R. Binet, A. Maounounen-Laasri, A. Lin, T. S. Hammack, and J. A. Kase, “Detection of five Shiga toxin-producing Escherichia coli genes with multiplex PCR,” Food Microbiol., vol. 40, 2014.[14] B. Li, H. Liu, and W. Wang, “Multiplex real-time PCR assay for detection of Escherichia coli O157 : H7 and screening for non-O157 Shiga toxin-producing E . coli,” pp. 1–13, 2017.[15] V. Barwick and S. Wood, “Achieving metrological traceability in chemical and bioanalytical measurement,” J. Anal. At. Spectrom., vol. 25, no. 6, pp. 785–799, 2010.[16] L. Wang et al., “Development of a Reference Standard of Escherichia coli DNA for Residual DNA Determination in China,” vol. 8, no. 9, pp. 1–6, 2013.[17] P. Van Iwaarden et al., “Certification of a Reference Material of Purified Genomic DNA from Escherichia Coli O157 Certified Reference Material IRMM-449,” 2006.[18] A. S. Devonshire, R. Elaswarapu, and C. A. Foy, “Applicability of RNA standards for evaluating RT-qPCR assays and platforms,” BMC Genomics, vol. 12, p. 10, Feb. 2011.[19] American Type Culture Collection (ATCC), “Certified Reference Materials (CRMs).” [Online]. Available: https://www.atcc.org/en/Standards/Standards_Programs/Certified_Reference_Materials_CRMs.aspx. [Accessed: 13-Jan-2020].[20] P. Corbisier et al., “CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue,” Metrologia, vol. 49, no. 1A, pp. 08002–08002, 2012.[21] S. Bhat and K. R. Emslie, “Digital polymerase chain reaction for characterisation of DNA reference materials,” Biomol. Detect. Quantif., vol. 10, pp. 47–49, 2016.[22] Proexport, “Programa de transformación productiva,” El priódico las oportunidades, pp. 1–24, 2012.[23] G. G. Moy and Y. Motarjemi, Public Health Measures: International Standards and Harmonization of Food Safety Legislation BT - Encyclopedia of Food Safety, vol. 4. Elsevier Ltd., 2014.[24] INS (Instituto Nacional de Salud), “Protocolo de vigilancia y control de enfermedades transmitidas por alimentos,” Bogotá D.C., 2011.[25] Ministerio de Salud y Protección Social, Resolución 4393 de 1991. Colombia, 1991, p. 3.[26] INVIMA, “Parametros microbiologicos de alimentos.,” 2011. .[27] Ministerio de Seguridad en Alimentos y Fármacos, “Food Code Corea Article 5.”[28] C. U. Europea, Reglamento (CE) n o 2073/2005 de la Comisión, de 15 de noviembre de 2005 , relativo a los criterios microbiológicos aplicables a los productos alimenticios (Texto pertinente a efectos del EEE), vol. 48. 2005, pp. 1–33.[29] INVIMA, “Portafolio de servicios,” 2015.[30] W. H. Organization, “Estimaciones de la OMS sobre la carga mundial de enfermedades de transmisión alimentaria,” World Heal. Organ., vol. 14, p. 2, 2015.[31] Jaime Alberto Guerrero, “Enfermedades Transmitidas por alimentos. PROTOCOLO DE VIGILANCIA EN SALUD PUBLICA,” Inst. Nac. Salud, pp. 3–4, 2016.[32] Instituto Nacional de Salud (INS), BES Semana epidemiológica 52 23 al 29 de Diciembre de 2018. 2018.[33] Organización Mundial de la Salud (OMS), “Riesgos microbiológicos.” [Online]. Available: https://www.who.int/foodsafety/areas_work/microbiological-risks/es/. [Accessed: 13-Jan-2020].[34] A. H. Havelaar et al., “World Health Organization Global Estimates and Regional Comparisons of the Burden of Foodborne Disease in 2010.”[35] J. B. Kaper, J. P. Nataro, and H. L. T. Mobley, “Pathogenic Escherichia coli,” Nat. Rev. Microbiol., vol. 2, no. 2, pp. 123–140, 2004.[36] FAO, “Escherichia coli,” Bol. enfermedades Transfront. los Anim., p. 39, 2011.[37] Foodborne disease burden epidemiology reference group, WHO estimates of the Global Burden of Foodborne Disease. 2015.[38] Y. Puig Peña, V. Leyva Castillo, N. Apórtela López, N. Campos González, Y. Frerer Marquez, and P. Soto Rodriguez, “Serogrupos y resistencia antimicrobiana de cepas de escherichia coli aisladas en alimentos procedentes de brotes de enfermedades diarreicas,” Rev. Cuba. Aliment. y Nutr., vol. 2, pp. 161–172, 2014.[39] F. Molina, E. López-acedo, R. Tabla, I. Roa, A. Gómez, and J. E. Rebollo, “Improved detection of Escherichia coli and coliform bacteria by multiplex PCR,” ???, pp. 1–9, 2015.[40] J. Y. L. J. W. Y. and C. J. Hovde, “A Brief Overview of Escherichia coli O157:H7 and Its Plasmid O157,” J Microbiol Biotechnol., vol. 20, no. 1, pp. 5–14, 2013.[41] C. Farrokh et al., “Review of Shiga-toxin-producing Escherichia coli (STEC) and their significance in dairy production,” Int. J. Food Microbiol., vol. 162, no. 2, pp. 190–212, 2013.[42] ISO, ISO 16649 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of b-glucuronidase-positive Escherichia coli. 2001.[43] ISO, “ISO 16649-2 Horizontal method for the enumeration of b-glucuronidase-positive Escherichia coli Part 2,” 2001.[44] A. Camacho, M. Giles, A. Ortegón, M. Palao, B. Serrano, and O. Velázquez, “Preparación y dilución de muestras de alimentos para su análisis microbiológico,” p. 9, 2009[45] N. Da Silva, M. H. Taniwaki, V. C. Junqueira, N. Silveira, M. da Silda do Nascimento, and R. Romeiro Gomes, Microbiological examination methods of food and water : a laboratory manual. 2013.[46] ISO, ISO 16654:2001. Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Escherichia coli 0157, no. 1. 2001.[47] International Organization for Standardization (ISO), “ISO 13136:2012 - Microbiology of food and animal feed — Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens — Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determi.” [Online]. Available: https://www.iso.org/standard/53328.html. [Accessed: 13-Jan-2020].[48] C. A. Baker, P. M. Rubinelli, S. H. Park, F. Carbonero, and S. C. Ricke, “Shiga toxin-producing Escherichia coli in food: Incidence, ecology, and detection strategies,” Food Control, vol. 59. p. 12, 2016.[49] S. Henson and R. Loader, “Barriers to agricultural exports from developing countries: The role of sanitary and phytosanitary requirements,” World Dev., vol. 29, no. 1, pp. 85–102, 2001.[50] European Comission, “RASFF - Food and Feed Safety Alerts - European Commission.” [Online]. Available: http://ec.europa.eu/food/safety/rasff_en. [Accessed: 25-Apr-2017].[51] L. E. Forero, “Regulación SPS de Alimentos para exportación a los Estados Unidos,” 2013.[52] European comissión, “RASFF Portal,” 2020. [Online]. Available: https://webgate.ec.europa.eu/rasff-window/portal/?event=searchResultList. [Accessed: 13-Jan-2020].[53] Ministerio de Salud y protección Social, “Resolucion 3929 de 2013 Requerimientos microbiológicos jugos.” 2013.[54] M. Mangal, S. Bansal, S. K. Sharma, and R. K. Gupta, “Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety,” Crit. Rev. Food Sci. Nutr., vol. 56, no. 9, pp. 1568–1584, Jul. 2016.[55] T. Kuchta et al., “A decade with nucleic acid-based microbiological methods in safety control of foods,” Lett. Appl. Microbiol., vol. 59, no. 3, pp. 263–271, 2014.[56] ISO, “ISO 4833-1 Horizontal method for the enumeration of microorganism,” vol. 44, no. 0, 2013.[57] P. K. Mandal, A. K. Biswas, K. Choi, and U. K. Pal, “Methods for Rapid Detection of Foodborne Pathogens: An Overview,” American Journal of Food Technology, vol. 6, no. 2. pp. 87–102, 2011.[58] G. Bou, A. Fernández-Olmos, C. García, J. A. Sáez-Nieto, and S. Valdezate, “Métodos de identificación bacteriana en el laboratorio de microbiología,” Enferm. Infecc. Microbiol. Clin., vol. 29, no. 8, pp. 601–608, 2011.[59] G. López-Campos, J. V. Martínez-Suárez, M. Aguado-Urda, and V. López-Alonso, “Microarray Detection and Characterization of Bacterial Foodborne Pathogens,” Food, Heal. Nutr., pp. 13–33, 2012.[60] J. W.-F. Law, N.-S. Ab Mutalib, K.-G. Chan, and L.-H. Lee, “Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations.,” Front. Microbiol., vol. 5, no. January, p. 770, 2015.[61] R. L. Bell, K. G. Jarvis, A. R. Ottesen, M. A. Mcfarland, and E. W. Brown, “Recent and emerging innovations in Salmonella detection: A food and environmental perspective,” Microb. Biotechnol., vol. 9, no. 3, pp. 279–292, 2016.[62] S. Bonetta et al., “Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay,” Environ. Sci. Pollut. Res., vol. 23, no. 15, pp. 15302–15309, 2016.[63] N. González-Escalona, E. W. Brown, and G. Zhang, “Development and evaluation of a multiplex real-time PCR (qPCR) assay targeting ttrRSBCA locus and invA gene for accurate detection of Salmonella spp. in fresh produce and eggs,” Food Res. Int., vol. 48, no. 1, pp. 202–208, 2012.[64] A. Rohde et al., “Overview of validated alternative methods for the detection of foodborne bacterial pathogens,” Trends Food Sci. Technol., vol. 62, pp. 113–118, 2017.[65] Z. P. Guan, Y. Jiang, F. Gao, L. Zhang, G. H. Zhou, and Z. J. Guan, “Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR,” Eur. Food Res. Technol., vol. 237, no. 4, pp. 627–637, 2013.[66] M. Varshney, L. Yang, X.-L. Su, and Y. Li, “Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157:H7 in ground beef.,” J. Food Prot., vol. 68, no. 9, pp. 1804–11, 2005.[67] V. Velusamy, K. Arshak, O. Korostynska, K. Oliwa, and C. Adley, “An overview of foodborne pathogen detection: In the perspective of biosensors,” Biotechnol. Adv., vol. 28, no. 2, pp. 232–254, 2010.[68] M. Pohanka, P. Skládal, and O. Pavliš, “Label‐Free Piezoelectric Immunosensor for Rapid Assay of Escherichia coli,” J. Immunoass. Immunochem., vol. 29, no. 1, pp. 70–79, 2007.[69] M. Vaz-Velho, G. Duarte, and P. Gibbs, “Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish,” J. Microbiol. Methods, vol. 40, no. 2, pp. 147–151, 2000.[70] D. Emerson, L. Agulto, H. Liu, and L. Liu, “Identifying and Characterizing Bacteria in an Era of Genomics and Proteomics,” Bioscience, vol. 58, no. 10, p. 925, 2008.[71] T. C. Chiu, “Recent advances in bacteria identification by matrix-assisted laser desorption/ionization mass spectrometry using nanomaterials as affinity probes,” Int. J. Mol. Sci., vol. 15, no. 5, pp. 7266–7280, 2014.[72] P.-E. Fournier, M. Drancourt, P. Colson, J.-M. Rolain, B. La Scola, and D. Raoult, “Modern clinical microbiology: new challenges and solutions,” Nat. Rev. Microbiol., vol. 11, no. 8, pp. 574–585, 2013.[73] N. T. Salihah, M. M. Hossain, H. Lubis, and M. U. Ahmed, “Trends and advances in food analysis by real-time polymerase chain reaction,” J. Food Sci. Technol., vol. 53, no. 5, pp. 2196–2209, 2016.[74] INVIMA, “Portafolio de servicios INVIMA,” pp. 1–7, 2018.[75] T. González Flores and R. A. Rojas Herrera, “Enfermedades transmitidas por alimentos y PCR: Prevención y diagnóstico,” Salud Publica Mex., vol. 47, no. 5, pp. 388–390, 2005.[76] M. T. Rahman, M. S. Uddin, R. Sultana, A. Moue, and M. Setu, “Polymerase Chain Reaction (PCR): A Short Review,” Anwer Khan Mod. Med. Coll. J., vol. 4, no. 1, pp. 30–36, 2013.[77] H. Ringuet et al., “hsp65 Sequencing for identification of rapidly growing mycobacteria,” J. Clin. Microbiol., vol. 37, no. 3, pp. 852–857, 1999.[78] G. W. Payne, P. Vandamme, S. H. Morgan, J. J. Lipuma, and T. Coenye, “Development of a recA gene-based identification approach for the entire Burkholderia genus,” Appl. Environ. Microbiol., vol. 71, no. 7, pp. 3917–3927, 2005.[79] L. Dong, Y. Meng, Z. Sui, J. Wang, L. Wu, and B. Fu, “Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.,” Sci. Rep., vol. 5, 2015.[80] S. Dhanasekaran, T. M. Doherty, and J. Kenneth, “Comparison of different standards for real-time PCR-based absolute quantification,” J. Immunol. Methods, vol. 354, no. 1–2, pp. 34–39, 2010.[81] S. Bhat and K. R. Emslie, “Digital polymerase chain reaction for characterization of DNA reference materials,” Biomol. Detect. Quantif., pp. 3–5, 2016.[82] L. Tamay de Dios, C. Ibarra, and C. Velasquillo, “Fundamentos de la reacción en cadena de la polimerasa (PCR) y de la PCR en tiempo real,” Learn. Discip. ICLS 2010 Conf. Proc. - 9th Int. Conf. Learn. Sci., vol. 12, pp. 70–78, 2013.[83] M. Arya, I. S. Shergill, M. Williamson, L. Gommersall, N. Arya, and H. R. H. Patel, “Basic principles of real-time quantitative PCR,” Expert Rev. Mol. Diagn., vol. 5, no. 2, pp. 209–219, 2005.[84] Y. Li, X. Zhou, and D. Ye, “Molecular beacons: An optimal multifunctional biological probe,” Biochem. Biophys. Res. Commun., vol. 373, no. 4, pp. 457–461, 2008.[85] E. Omiccioli, G. Amagliani, G. Brandi, and M. Magnani, “A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk,” Food Microbiol., vol. 26, no. 6, pp. 615–622, 2009.[86] S. H. Liming and A. A. Bhagwat, “Application of a molecular beacon - Real-time PCR technology to detect Salmonella species contaminating fruits and vegetables,” Int. J. Food Microbiol., vol. 95, no. 2, pp. 177–187, 2004.[87] S. Perelle, F. Dilasser, J. Grout, and P. Fach, “Detection by 5′-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world’s most frequent clinical cases,” Mol. Cell. Probes, vol. 18, no. 3, pp. 185–192, 2004.[88] L. W. Noll et al., “A Four-Plex Real-Time PCR Assay, Based on rfb E, stx 1, stx 2, and eae Genes, for the Detection and Quantification of Shiga Toxin–Producing Escherichia coli O157 in Cattle Feces,” Foodborne Pathog. Dis., vol. 12, no. 9, pp. 787–794, 2015.[89] G. A. Leotta et al., “Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga,” Rev. Argent. Microbiol., vol. 37, no. 1, pp. 1–10, 2005.[90] B. Vogelstein and K. W. Kinzler, “Digital PCR,” Genetics, vol. 96, no. August, p. 92369241, 1999.[91] A. A. Morley, “Digital PCR: A brief history,” Biomol. Detect. Quantif., vol. 1, no. 1, pp. 1–2, 2014.[92] J. F. Huggett, J. A. Garson, and A. S. Whale, “Digital PCR and Its Potential Application to Microbiology,” Mol. Microbiol. Diagnostic Princ. ans Pract., pp. 49–57, 2016.[93] M. Wang et al., “Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk,” Int. J. Food Microbiol., vol. 266, 2018.[94] J. F. Huggett, S. Cowen, and C. A. Foy, “Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool,” vol. 1, no. 61, pp. 79–88, 2015.[95] B. Verhaegen, K. De Reu, L. De Zutter, K. Verstraete, M. Heyndrickx, and E. Van Coillie, “Comparison of droplet digital PCR and qPCR for the quantification of shiga toxin-producing Escherichia coli in bovine feces,” Toxins (Basel)., vol. 8, no. 5, pp. 1–11, 2016.[96] R. Sanders, D. J. Mason, C. A. Foy, and J. F. Huggett, “Evaluation of Digital PCR for Absolute RNA Quantification,” PLoS One, vol. 8, no. 9, pp. 1–9, 2013.[97] D. G. Burke et al., “Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity,” Anal. Chem., vol. 85, no. 3, pp. 1657–1664, 2013.[98] R. Sanders, J. F. Huggett, C. A. Bushell, S. Cowen, D. J. Scott, and C. A. Foy, “Evaluation of digital PCR for absolute DNA quantification,” Anal. Chem., vol. 83, no. 17, pp. 6474–6484, 2011.[99] S. Bhat, N. Curach, T. Mostyn, G. S. Bains, K. R. Griffiths, and K. R. Emslie, “Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units,” Anal. Chem., vol. 82, no. 17, pp. 7185–7192, 2010.[100] Biomerieux, “Métodos Rápidos para el Métodos Rápidos para el control Microbiológico de Alimentos,” pp. 1–54, 2011.[101] G. Pohl and M. Shih Ie, “Principle and applications of digital PCR,” Expert Rev Mol Diagn, vol. 4, no. 1, pp. 41–47, 2004.[102] M. Boyer and J. Combrisson, “Analytical opportunities of quantitative polymerase chain reaction in dairy microbiology,” Int. Dairy J., vol. 30, no. 1, pp. 45–52, 2013.[103] G. Agustí, M. Fittipaldi, and F. Codony, “Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples,” Curr. Microbiol., vol. 75, no. 6, pp. 779–785, 2018.[104] S. Broeders et al., “Guidelines for validation of qualitative real-time PCR methods,” Trends Food Sci. Technol., vol. 37, no. 2, pp. 115–126, 2014.[105] J. F. Huggett, S. Cowen, and C. A. Foy, “Considerations for digital PCR as an accurate molecular diagnostic tool,” Clin. Chem., vol. 61, no. 1, pp. 79–88, 2015.[106] P. Truchado, M. I. Gil, T. Kostic, and A. Allende, “Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production,” Food Control, vol. 62, pp. 150–156, 2016.[107] E. T. Gensberg, M. Polt, M. Konrad-k, P. Kinner, A. Sessitsch, and T. Kostic, “Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality,” Water Res., vol. 67, no. 0, pp. 367–376, 2014.[108] B. Li, Z. Hu, and C. a Elkins, “Detection of live Escherichia coli O157:H7 cells by PMA-qPCR.,” J. Vis. Exp., no. 84, p. e50967, 2014.[109] M. Chapela, A. Garrido-Maestu, and A. G. Cabado, “Detection of foodborne pathogens by qPCR : A practical approach for food industry applications,” Cogent Food Agric., vol. 1, no. 1, pp. 1–19, 2015.[110] J. Pavšič et al., “Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA,” Anal. Bioanal. Chem., 2017.[111] H. J. He, J. L. Almeida, S. P. Lund, C. R. Steffen, S. Choquette, and K. D. Cole, “Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements,” Biomol. Detect. Quantif., vol. 8, pp. 1–8, 2016.[112] C. Floren, I. Wiedemann, B. Brenig, E. Schütz, and J. Beck, “Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR),” Food Chem., vol. 173, pp. 1054–1058, 2015.[113] M. Milavec, K. Gruden, and Z. Jana, “Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR,” PLoS One, vol. 8, no. 5, 2013.[114] H. White et al., “A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR,” Leukemia, vol. 29, no. 2, pp. 369–376, 2015.[115] C. M. Hindson et al., “Absolute quantification by droplet digital PCR versus analog real-time PCR,” Nat. Methods, vol. 10, no. 10, pp. 1003–1005, 2013.[116] M. C. Kline, E. L. Romsos, and D. L. Duewer, “Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets,” Anal. Chem., p. acs.analchem.5b03692, 2016.[117] J. Pavšič, J. Žel, and M. Milavec, “Digital PCR for direct quantification of viruses without DNA extraction,” Anal. Bioanal. Chem., vol. 408, no. 1, pp. 67–75, 2016.[118] L. Gerdes, A. Iwobi, U. Busch, and S. Pecoraro, “Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms,” Biomol. Detect. Quantif., vol. 7, pp. 9–20, 2016.[119] M. Sivaganesan, M. Varma, S. Siefring, and R. Haugland, “Quantification of plasmid DNA standards for U.S. EPA fecal indicator bacteria qPCR methods by droplet digital PCR analysis,” J. Microbiol. Methods, vol. 152, pp. 135–142, Sep. 2018.[120] D. L. Duewer, M. C. Kline, E. L. Romsos, and B. Toman, “Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter,” Anal. Bioanal. Chem., vol. 410, no. 12, 2018.[121] J. F. Huggett et al., “The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.,” Clin. Chem., vol. 59, no. 6, pp. 892–902, Jun. 2013.[122] T. Soejima, J. I. Minami, T. Yaeshima, and K. Iwatsuki, “An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk,” Appl. Microbiol. Biotechnol., vol. 95, no. 2, pp. 485–497, 2012.[123] A. Rompré, P. Servais, J. Baudart, M. R. De-Roubin, and P. Laurent, “Detection and enumeration of coliforms in drinking water: Current methods and emerging approaches,” J. Microbiol. Methods, vol. 49, no. 1, pp. 31–54, 2002.[124] Adria Developpement, “Validation of alternative analytical methods Application in food microbiology Summary report EN ISO 16140 validation study of the DuPont TM BAX detection of Salmonella spp in meat products , egg,” 2015.[125] M. L. Camaró-Sala, R. Martínez-García, P. Olmos-Martínez, V. Catalá-Cuenca, M. D. Ocete-Mochón, and C. Gimeno-Cardona, “Validación y verificación analítica de los métodos microbiológicos,” Enfermedades Infecc. y Microbiol. Clin. Monogr., vol. 33, no. 7, pp. e31–e36, 2015.[126] AFNOR, “E.coli O157 methods.” [Online]. Available: https://nf-validation.afnor.org/en/food-industry/e-coli-o157/. [Accessed: 14-Jan-2020].[127] AOAC, “Official Methods of Analysis.” [Online]. Available: http://www.eoma.aoac.org/. [Accessed: 14-Jan-2020].[128] AFNOR, “Validation of alternative analytical methods Application in food microbiology Molecular Detection Assay E. coli O157 (including H7) for the detection of Escherichia coli O157 (including H7) in raw beef meat, raw dairy products, raw fruits & vegetables,” 2016.[129] ISHA, “NF VALIDATION 16140 TM AFNOR CERTIFICATION VALIDATION OF THE METHOD ADIAFOOD Escherichia coli O157 : H7 method ( single well ) For the detection of Escherichia coli O157 : H7 SUMMARY REPORT ‐ SEPTEMBER 2015 – V1 Expert laboratory : ISHA 25 avenue de la Ré,” 2015.[130] Adria Developpement, “Rapport de synthèse Reconduction de la validation EN ISO 16140 de la méthode Bax® E. coli O157:H7 MP,” 2012.[131] Adria Developpement, “Validation study according to the EN ISO 16140 standard Summary report EN ISO 16140 validation of the DuPont TM BAX ® Real-Time PCR Assay for E . coli O157 : H7 method in raw beef meats and raw vegetables,” 2014.[132] AFNOR, “Validation of alternative analytical methods Application in food microbiology validation study of the GeneDisc® method for the simultaneous detection of Salmonella spp. and Escherichia coli O157:H7-Detection of Escherichia coli O157:H7,” 2017.[133] Adria Developpement, “Rapport de synthèse Validation EN ISO 16140 de la méthode Méthode iQ-Check Eschericia coli O157:H7,” 2012.[134] Adria Developpement, “Summary report EN ISO 16140 validation study of the MicroSEQ ® Escherichia coli O157 : H7 method for the detection of Escherichia coli O157 : H7 in raw beef meats and raw vegetables,” 2015.[135] S. Pillet et al., “Comparative Evaluation of Six Commercialized Multiplex PCR Kits for the Diagnosis of Respiratory Infections,” PLoS One, vol. 8, no. 8, 2013.[136] P.-Y. Cheung, C. W. Chan, W. Wong, T. L. Cheung, and K. M. Kam, “Evaluation of two real-time polymerase chain reaction pathogen detection kits for Salmonella spp. in food,” Lett. Appl. Microbiol., vol. 39, no. 6, pp. 509–515, Dec. 2004.[137] EURACHEM, “La Adecuación al Uso de los Métodos Analíticos.” 2016, p. 66, 2016.[138] JCGM (Comité Conjunto para las Guías en Metrología), Vocabulario Internacional de Metrología. 2008.[139] International Organization for Standardization, ISO 17511 INvitro diagnostic medical devices-Measurement of quantities in biological samples - Metrological traceability of values assigned to calibrators and control materials, vol. 2005. 2006.[140] M. Thompson, S. L. R. Ellison, and R. Wood, “Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC Technical Report),” Int. Union Pure Appl. Chem., vol. 74, no. 5, pp. 835–855, 2002.[141] S. L. R. Ellison, M. Rosslein, A. Williams, L. A. Konopelko, and A. V. Garmash, “EURACHEM/CITAC Guide: Quantifying Uncertainty in Analytical Measurement,” Journal of Analytical Chemistry, vol. 58, no. 2. European Federation of National Associations of Analytical Laboratories, p. 191, 2003.[142] BIPM, International vocabulary of metrology - Basic and general concepts and associated terms (VIM). 2012.[143] EURACHEM and B. King, “The selection and use of reference materials,” 2002.[144] “ISO/Guide 33:2015(en), Reference materials — Good practice in using reference materials.” [Online]. Available: https://www.iso.org/obp/ui/#iso:std:iso:guide:33:ed-3:v1:en. [Accessed: 14-Jan-2020].[145] W. R. Wolf, “History of reference materials for food and nutrition metrology: As represented in the series of BERM symposia,” Anal. Bioanal. Chem., vol. 397, no. 2, pp. 413–421, 2010.[146] Societa Italiana di Fisica, Metrology: from Physics Fundamentals to Quality of Life. 2017.[147] NIST, “Standard Reference Materials.” [Online]. Available: https://www.nist.gov/srm. [Accessed: 14-Jan-2020].[148] “Certified Reference Materials catalogue of the JRC.” [Online]. Available: https://crm.jrc.ec.europa.eu/. [Accessed: 14-Jan-2020].[149] WHO (World Health Organization), “WHO | International reference materials.” [Online]. Available: https://www.who.int/bloodproducts/ref_materials/en/. [Accessed: 14-Jan-2020].[150] BIPM, “Database of higher-order reference materials, measurement methods/procedures and services.” [Online]. Available: https://www.bipm.org/jctlm/. [Accessed: 13-Jan-2020].[151] L. Deprez et al., “Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material,” Biomol. Detect. Quantif., vol. 9, pp. 29–39, 2016.[152] P. Van Iwaarden et al., “Certification of a Reference Material of Purified Genomic DNA from Escherichia Coli O157 Certified Reference Material IRMM-449,” vol. 157, no. Edl 933.[153] J. R. C. JRC, Certification of Reference Materials of Maize Seed Powder containing Genetically Modified MON 810 Maize Certified Reference Materials ERM ® -BF413k. .[154] P. Corbisier, “CERTIFICATION REPORT A set of three plasmid DNA calibration solutions bearing a porcine- specific DNA fragment A set of three plasmid DNA calibration solutions bearing a porcine-specific DNA fragment,” 2015.[155] P. Corbisier et al., “DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials,” Anal. Bioanal. Chem., vol. 407, no. 7, pp. 1831–1840, 2015.[156] Zeptometrix Corporation, “Escherichia coli O157:H7; EDL933, Genomic DNA,” 2014, p. 801622.[157] International Organization for Standardization (ISO), “ISO/Guide 30:2015(en), Reference materials — Selected terms and definitions.” [Online]. Available: https://www.iso.org/obp/ui/#iso:std:iso:guide:30:ed-3:v1:en. [Accessed: 14-Jan-2020].[158] W. A. Schmid and R. J. Lazos Martínez, “Guide to estimate the measurement uncertainty,” Natl. Cent. Metrol. Mex., p. 27, 2000.[159] T. P. J. Linsinger, J. Pauwels, A. Lamberty, H. G. Schimmel, A. M. H. Van Der Veen, and L. Siekmann, “Estimating the uncertainty of stability for matrix CRMs,” Anal. Bioanal. Chem., vol. 370, no. 2–3, pp. 183–188, 2001.[160] A. M. H. Van Der Veen, T. P. J. Linsinger, H. Schimmel, A. Lamberty, and J. Pauwels, “Uncertainty calculations in the certification of reference materials 4. Characterisation and certification,” Accredit. Qual. Assur., vol. 6, no. 7, pp. 290–294, 2001.[161] I. O. for S. ISO, “ISO 17034:2016(es), Requisitos generales para la competencia de los productores de materiales de referencia.” [Online]. Available: https://www.iso.org/obp/ui#iso:std:iso:17034:ed-1:v1:es. [Accessed: 14-Jan-2020].[162] World Health Organization, “Recommendations for the preparation, characterization and establishment of international and other biological reference standards,” WHO Tech. Rep. Ser. 932, vol. 2, no. 932, pp. 73–131, 2006.[163] International Organization for Standardization (ISO), “ISO - ISO Guide 35:2017 - Reference materials — Guidance for characterization and assessment of homogeneity and stability.” [Online]. Available: https://www.iso.org/standard/60281.html. [Accessed: 13-Jan-2020].[164] E. D. F. Guimarães, E. C. P. Do Rego, H. C. M. Cunha, J. M. Rodrigues, J. D. F. Villar, and V. S. Da Cunha, “Homogeneity study for certification of a candidate reference material for polycyclic aromatic hydrocarbons,” 19th IMEKO World Congr. 2009, vol. 4, pp. 2377–2381, 2009.[165] T. P. J. Linsinger, J. Pauwels, A. M. H. Van Der Veen, H. Schimmel, and A. Lamberty, “Homogeneity and stability of reference materials,” Accredit. Qual. Assur., vol. 6, no. 1, pp. 20–25, Jan. 2001.[166] T. P. J. Linsinger, J. Pauwels, A. M. H. Van Der Veen, H. Schimmel, and A. Lamberty, “Homogeneity and stability of reference materials,” Accredit. Qual. Assur., vol. 6, no. 1, pp. 20–25, 2001.[167] A. Lamberty, H. Schimmel, and J. Pauwels, “The study of the stability of reference materials by isochronous measurements,” in Fresenius’ Journal of Analytical Chemistry, 1998, vol. 360, no. 3–4, pp. 359–361.[168] ISO Guide 35, “Reference materials -- General and statistical principles for certification.” International Organization for Standardization. Geneva, Suiza, 2006.[169] A. B. Košir et al., “Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR,” Anal. Bioanal. Chem., vol. 409, no. 28, pp. 6689–6697, 2017.[170] J. F. Huggett et al., “The digital MIQE guidelines: Minimum information for publication of quantitative digital PCR experiments,” Clin. Chem., vol. 59, no. 6, pp. 892–902, 2013.[171] K. Andersen and J. Merry, “Reference Materials,” vol. 18, no. 6, pp. 376–383, 1999.[172] E. Theres, M. Polt, M. Konrad-k, P. Kinner, A. Sessitsch, and T. Kosti, “Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality,” vol. 7, no. 0, pp. 367–376, 2014.[173] E. M. Nielsen and M. T. Andersen, “Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5 ′ Nuclease PCR Assay Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5Ј Nuclease PCR Assay,” J. Clin. Microbiol., vol. 41, no. 7, pp. 2884–2893, 2003.[174] I. O. for S. ISO, “ISO/TS 13136:2012 - Microbiology of food and animal feed -- Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens -- Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups,” 2012. [Online]. Available: https://www.iso.org/standard/53328.html. [Accessed: 21-Mar-2019].[175] Integrated DNA technologies, “How do I use the OligoAnalyzer tool to analyze possible hairpins and dimers formed by my oligo?” [Online]. Available: https://www.idtdna.com/pages/support/faqs/how-do-i-use-the-oligoanalyzer-tool-to-analyze-possible-hairpins-and-dimers-formed-by-my-oligo. [Accessed: 24-Sep-2019].[176] S. Das Mitrai et al., “Duplex PCR for specific detection of Escherichia coli and its differentiation from other Enterobacteriaceae,” Indian J. Anim. Sci., vol. 85, no. 8, pp. 16–19, 2015.[177] K. Horakova, H. Mlejnkova, and P. Mlejnek, “Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: Cytochrome bd complex, lactose permease, ??-d-glucuronidase, and ??-d-galactosidase,” J. Appl. Microbiol., vol. 105, no. 4, pp. 970–976, 2008.[178] B. Li and J. Chen, “Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157 : H7 Cells by Targeting Z3276 as a Genetic,” vol. 78, no. 15, pp. 5297–5304, 2012.[179] E. M. Nielsen and M. T. Andersen, “Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5 ′ Nuclease PCR Assay Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5Ј Nuclease PCR Assay,” J. Clin. Microbiol., vol. 41, no. 7, pp. 2884–2893, 2003.[180] E. Barbau-Piednoir, S. Bertrand, J. Mahillon, N. H. Roosens, and N. Botteldoorn, “SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels,” Appl. Microbiol. Biotechnol., vol. 97, no. 22, pp. 9811–9824, 2013.[181] BIORAD, “Real-time PCR: Applications Guide,” Bio-Rad Lab., pp. 2–84, 2006.[182] V. K. Singh, R. Govindarajan, S. Naik, and A. Kumar, “The effect of hairpin structure on PCR amplification efficiency.,” Mol Biol Today, vol. 1, no. 3, pp. 67–9, 2000.[183] A. Ud-Din and S. Wahid, “Relationship among Shigella spp. And enteroinvasive Escherichia coli (EIEC) and their differentiation,” Brazilian J. Microbiol., vol. 45, no. 4, pp. 1131–1138, 2014.[184] Bio-Rad, “Droplet Digital TM PCR Applications guide.”[185] Promega, “Assembly of Restriction Enzyme Digestions,” 2007.[186] W. Liang et al., “Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR,” Chem. Cent. J., vol. 10, no. 1, p. 55, 2016.[187] R. T. Hayden et al., “Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus,” J. Clin. Microbiol., vol. 51, no. 2, pp. 540–546, 2013.[188] M. E. Hunter, R. M. Dorazio, J. S. S. Butterfield, G. Meigs-Friend, L. G. Nico, and J. A. Ferrante, “Detection limits of quantitative and digital PCR assays and their influence in presence???absence surveys of environmental DNA,” Mol. Ecol. Resour., vol. 17, no. 2, pp. 221–229, 2017.[189] Thermo Scientific, “T042-TECHNICAL BULLETIN NanoDrop Spectrophotometers.”[190] C. Villamil, “Desarrollo de herramientas de aseguramiento metrológico para la identificación y cuantificación de Salmonella spp. por métodos basados en PCR,” Universidad Nacional de Colombia, 2019.[191] “ISO/Guide 31:2015(en), Reference materials — Contents of certificates, labels and accompanying documentation.” [Online]. Available: https://www.iso.org/obp/ui/#iso:std:iso:guide:31:ed-3:v1:en. [Accessed: 14-Jan-2020].[192] G. M.M., E. P., L. V., L. M. M., S. L. L., and A. M. L., “[Detection of diarrheagenic Escherichia coli in children from poor neighborhoods in Corrientes, Argentina],” Rev. Cubana Med. Trop., vol. 62, no. 1, pp. 42–47, 2010.[193] A. Paton and J. Paton, “Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex Enterohemorrhagic E . coli hlyA , rfb O111 , and Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stx 1 , stx 2 , eae,” J. Clin. Microbiol., vol. 36, no. 2, pp. 598–602, 1998.[194] B. China, V. Pirson, and J. Mainil, “Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes,” Appl. Environ. Microbiol., vol. 62, no. 9, pp. 3462–3465, 1996.[195] W. Liang et al., “Quantification of plasmid DNA reference materials for Shiga toxin ‑ producing Escherichia coli based on UV , HR ‑ ICP ‑ MS and digital PCR,” pp. 1–10, 2016.[196] D. R. Pollard, W. M. Johnson, H. Lior, S. D. Tyler, and K. R. Rozee, “Erratum: Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction (J. Clin. Microbiol., Volume 28, No. 3, P. 542),” J. Clin. Microbiol., vol. 28, no. 6, p. 1491, 1990.[197] Y. Hu, Q. Zhang, and J. C. Meitzler, “Rapid and sensitive detection of Escherichia coli O157 : H7 in bovine faeces by a multiplex PCR,” J. Appl. Microbiol., vol. 87, pp. 867–876, 1999.[198] V. Brusa, L. Galli, L. H. Linares, E. E. Ortega, J. P. Lirón, and G. A. Leotta, “Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat,” J. Microbiol. Methods, vol. 119, pp. 10–17, 2015.[199] K. A. Ziebell, S. C. Read, R. P. Johnson, and C. L. Gyles, “Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins,” Res. Microbiol., vol. 153, no. 5, pp. 289–300, 2002.[200] R. Gordillo, A. Rodríguez, M. L. Werning, E. Bermúdez, and M. Rodríguez, “Quantification of viable Escherichia coli O157:H7 in meat products by duplex real-time PCR assays.,” Meat Sci., vol. 96, no. 2, pp. 964–970, 2014.[201] E. C. Chern, S. Siefring, J. Paar, M. Doolittle, and R. A. Haugland, “Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes,” Lett. Appl. Microbiol., vol. 52, no. 3, pp. 298–306, 2011.[202] E. Frahm and U. Obst, “Application of the fluorogenic probe technique ( TaqMan PCR ) to the detection of Enterococcus spp . and Escherichia coli in water samples,” vol. 52, pp. 123–131, 2003.[203] J. N. Miller and J. C. Miller, Estadística y Quimiometría para Química Analítica, 4a edición. 2002.ORIGINAL1020752137_2020.pdf1020752137_2020.pdfapplication/pdf3716295https://repositorio.unal.edu.co/bitstream/unal/77923/1/1020752137_2020.pdf3f3ea0e0fb14ae39145a65346298e7f9MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-83991https://repositorio.unal.edu.co/bitstream/unal/77923/2/license.txt6f3f13b02594d02ad110b3ad534cd5dfMD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.unal.edu.co/bitstream/unal/77923/3/license_rdf217700a34da79ed616c2feb68d4c5e06MD53THUMBNAIL1020752137_2020.pdf.jpg1020752137_2020.pdf.jpgGenerated Thumbnailimage/jpeg5032https://repositorio.unal.edu.co/bitstream/unal/77923/4/1020752137_2020.pdf.jpg2382d0345f56f7ca44351475541664dfMD54unal/77923oai:repositorio.unal.edu.co:unal/779232023-07-23 23:03:58.304Repositorio Institucional Universidad Nacional de Colombiarepositorio_nal@unal.edu.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 |